Establishment of a novel in vitro transcription assay

RNA Polymerase II (Pol II) transcriptional recycling is an underappreciated mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. By combing our in vitro transcription assays with other experiments (e.g. in vivo dynamic ChIP-seq assays), we identified PAF1 complex components as drivers for transcription recycling, revealing a new layer in controlling Pol II-dependent transcription. Our findings also point to RNA Pol II transcription recycling as a mechanism that functions aberrantly in disease states such as cancer, and indicate the potential to target factors such as PAF1 that drive RNA Pol II recycling in cancer.